Methods for the diagnosis and treatment of endometriosis

ABSTRACT

The present invention relates to methods of detecting CD200L in a female subject from a secretory phase biological sample. Increased levels of CD200L have been determined to be associated with endometriosis. CD200L is therefore a useful biomarker for the diagnosis of endometriosis and a useful target for therapeutic intervention.

FIELD OF INVENTION

The present invention provides compositions and methods for thediagnosis, assessment, characterization and treatment of endometriosisin a subject in need thereof, based upon the expression level of abiomarker that is associated with endometriosis.

BACKGROUND OF THE INVENTION

Although menstrual tissue with blood is expelled primarily per vaginum,in 70-90% of women, some menstrual tissue refluxes back through thefallopian tubes into the peritoneal cavity and is thought to give riseto endometriosis, a concept originally proposed by Sampson (Clement,2007; Bokor et al., 2009; Li et al., 2014). Endometrial epithelium andstroma successfully implants on the peritoneal surface of the uterus,ovary, pouch of Douglas peritoneum and other peritoneal surfaces such asthe omentum (Clement, 2007; Bokor et al., 2009; Li et al., 2014).Successfully implanted peritoneal deposits of endometrial cellsstimulated by estrogen can grow, cause local inflammation with releaseof Th1 cytokines that inhibit sperm-oocyte interactions (even ininfertile patients where endometriotic implants are not visible), andeventually, may lead to fibrosis as the inflammatory/healing processburns out (Clement, 2007; Kalu et al., 2007). Although this process isreminiscent of surface spread of malignant tumor cells, endometrioticdeposits are not usually invasive and are not usually found in lymphnodes. Endometriotic deposits have been occasionally seen in veins andskin, deposits in the liver and lung are extremely rare absent malignanttransformation (Clement, 2007; Bokor et al., 2009; Yu et al., 2013; Liuet al., 2015). However, endometriotic deposits can develop in abdominalscars, and along needle biopsy tracks, supporting the idea that theability of endometrial cells to establish extrauterine deposits is aninherent property of the endometrial cells (Wolf and Singh, 1989).Ridley and Edwards have reported successful autologous menstrual bloodcell implantation in the suprapubic area adjacent to peritoneal fasciawith ⅛ women developing an endometrioma and 2/8 having a foreignbody/scarring reaction (Ridley and Edwards, 1958). Human endometrium aswell as endometriosis tissue has been successfully xenotransplanted intoimmunodeficient nu/nu mice with>80% success and no evidence thatproliferative and secretory phase endometrium were different in theirability to implant (Zamah et al., 1984). A similar result was obtainedwith autologous endometrium in monkeys and also using menstrually shedendometrial cells but the success rate in establishing endometriosis wasless than in the xenotransplant mouse model (Zamah et al., 1984; TeLinde and Scott, 1950; Scott and Te Linde, 1954).

The implantation efficiency of endometrial cells entering into theperitoneal cavity at the time of menstruation is estimated to be only11-14% in the 90% of women with retrograde menstruation (Clement, 2007;Bokor et al., 2009; Li et al., 2014). Many factors have been suggestedto play a role in the implantation efficiency. In a mouse model, alarger dose of endometrial cells plus administration of estrogen hasbeen shown to be critical (Somigliana et al., 1999). Estrogen is alsopermissive for human endometriosis (Tirado-Gonzalez et al., 2010; Xu etal., 2013; Mei et al., 2015). Gargett et al., (2014) have postulatedthat reflux of endometrial stem cells into the peritoneal cavity occursin 5% of female neonates, and later in life (albeit sometimes beforeadolescence) and these stem cells become activated and cause early onsetendometriosis. In adult women, higher frequencies of endometrial stemcells in menstrual blood and peritoneal fluid may similarly predisposeto endometriosis (Gargette et al., 2014).

Notwithstanding the inherent autotransplantability and dose ofendometrial cells delivered to the peritoneal cavity (the seed), thereappears to be critically important co-factors required to ensure theperitoneal environment (the soil) is hospitable. For example, NK cellsuppression facilitates grafting, whereas cytokines such as IL-2 andIL-12 that activate NK cells have been shown to be inhibitory(Somigliana et al., 1999). In endometriosis, there is suppression ofinnate effector cells such as cytolytic CD56⁺CD16⁺ NK cells (Vigano etal., 1991; Jones et al., 1996; Berbic and Fraser, 2011) and based onanimal model data, suppression of NK cells appears important forestablishment of ectopic implants (Somigliana et al., 1999).Upregulation of IDO in endometrial stromal cells promotes growth andaugments production of IL-33 which renders macrophages ‘tolerant’ (Meiet al., 2012, 2013). IDO also degrades tryptophan into molecules thatenhance the generation of regulatory T cells (Tregs), that suppressrejection, suppress NK cells, and render macrophages ‘tolerant’ (Clark,2016a). Treg suppressor activity similar to what is required for normalembryo implantation in pregnancy occurs in endometriotic deposits andoriginating endometrium, along with production of IDO that promotesTregs, and recruitment of ‘tolerant’ myeloid-derived suppressormacrophage-type cells (Berbic et al., 2010; Berbic and Fraser, 2011;Clark, 2016a). The macrophages in ectopic implants produce growthfactors as well as IDO which stimulates macrophages to produce theimmunosuppressive cytokine IL-10 and fibrogenic cytokine TGF-β, and alsostimulates endometriotic cell growth and invasion (Mei et al., 2012,2015).

Treg cells are present in ectopic and eutopic endometrium ofendometriosis patients and are proposed to play a key role in theestablishment and progression of ectopic implants (Basta et al., 2010;Berbic et al., 2010; Berbic and Fraser, 2011). The role of Tregs cellsis supported by studies of experimental endometriosis in mice, and mayact in a number of ways (Budiu et al., 2009; Clark and Gorczynski, 2013;Woidacki et al., 2015). Tregs may also stimulate, directly orindirectly, angiogenic CD56^(bright)CD16⁻ NK cells that home toinflammatory sites where they promote angiogenesis (albeit only 5% ofleukocytes in endometriosis tissue are CD56⁺CD16⁺), angiogenicIDO-producing macrophages, angiogenic neutrophils and angiogenic mastcells (Dalbeh et al., 2004; Berbic and Fraser, 2011; Jetten et al.,2014; Clark, 2016a). Thus, a variety of processes dominated by Tregcells make the “soil” (i.e. peritoneum) favorable for refluxed shedendometrium to implant and grow.

One property of eutopic endometrium proposed to make it more likely toimplant and grow when refluxed into the peritoneal cavity at the time ofmenstruation is reduced expression of IL-18. IL-18 normally promotes thecytolytic interferon-y-producing type of cytolytic NK cells (Luo et al.,2006). IL-18 is another pro-inflammatory cytokine which may besuppressed in eutopic endometriosis along with other Th1 cytokines byTreg cells.

The CD200 tolerance signaling molecule is a glycoprotein that acts bybinding to CD200 receptors which possess an intracellular signalingtail. Binding on dendritic cells promotes IDO production and promotesTreg generation, Binding to macrophages promotes IDO-production thatenhances Treg generation and converts proinflammatory M1-typemacrophages to ‘tolerogenic’ type M2 macrophages (Clark, 2016a). Tregs,as well as CD200 acting directly on CD200R, can suppress cytolytic NKcells that can reject deposition and survival of damaged/sloughed tissue(Clark and Gorczynski, 2013; Clark, 2016a, 2016b). CD200 can alsodirectly suppress mast cells. Tregs are present in endometriosisdeposits. It has been suggested by Berbic et al., (2010) and Berbic andFraser (2011) that Treg cell levels may be higher in the pre-menstrual(secretory phase) endometrium of women who have endometriosis, althoughtheir identification of Tregs relied solely on expression ofintracellular foxp3 which alone is now thought to be insufficient toidentify Tregs in humans. CD200 is present in the trophoblast of earlyhuman embryos and plays an important role in ensuring pregnancy successby activating Tregs and suppressing harmful inflammation and NK cellcytotoxicity. CD200 is released in a bioactive soluble form (sCD200) andcan act at a distance from the cell producing it (Wong et al., 2016).

However, eutopic endometrial parameters sufficient to predictendometriosis have not been identified to date. Berbic et al. (2010)reported more Foxp3⁺ cells in the secretory phase endometrium ofendometriosis patients, but with 21 controls and 25 patients, thestandard deviation was so large as to indicate significant overlap, andonly ⅓ of endometriosis patients had counts exceeding the 95% confidencerange of normal non-endometriosis control patients. Thus, Foxp3 celldensity in an individual would be predictive for only ⅓ of endometriosispatients. It should be noted that in humans, Foxp3 is expressed byactivated T cells as well as by Treg cells, so Treg cell identificationrequires additional markers (Allan et al., 2007). With respect to IL-18,the levels in eutopic endometrium in endometriosis patientssignificantly overlap the normal control range (Luo et al., 2006), sothe predictive value of IL-18 for endometriosis is no better than Foxp3.It has been mentioned that high IDO levels in endometrial stromal cellsthat induces production of IL-33 that promotes inflammation by bindingto T1/ST2 receptors. Peritoneal IL-33 levels in early stageendometriosis are not different from the levels in non-endometriosiscontrols (Mbarik et al., 2015). IDO levels have only been quantified inendometriosis tissue and not in normal control tissue. Activated NKcells are implicated in preventing endometriosis, however, endometriosiscan occur in spite of increased peritoneal NK activity (Somigliana etal., 1999; Wu et al., 2000). The susceptibility to suppression of NKcells in different individual may also vary (Somigliana et al., 2001).

This raises the question of genetic factors that might determine risk ofendometriosis (Vigano et al., 2007). Genetic factors contributeincreased risk of endometriosis based on twin studies (Treloar et al.,1999), but relevance for CD200 and CD200R expression is unknown. Geneexpression in endometriomas differs from eutopic endometrium, which isexplainable by inflammation at ectopic sites (Matsuzaki et al., 2006),and is complicated by the fact that peritoneal inflammation inendometriosis patients alters gene expression in eutopic endometrium(Brosens et al., 2012). None of the differences between eutopicendometrium in normal patients versus endometriosis patients can beconsidered a reliable indicator of what properties of eutopicendometrium predicted likelihood of developing endometriosis (Brosens etal., 2012).

Accordingly, it would be desirable to develop a novel method ofdiagnosing and treating endometriosis.

SUMMARY OF THE INVENTION

The present invention describes the finding that increased levels ofCD200L are associated with endometriosis. CD200L is therefore a usefulbiomarker for the diagnosis of endometriosis and a useful target fortherapeutic intervention.

In one aspect of the invention, a method of detecting CD200L in asecretory phase endometrial biological sample from a female mammal orsubject is provided. The method comprises: i) obtaining the secretoryphase biological sample from the subject, and ii) detecting whether ornot CD200L is present in the biological sample by contacting the samplewith an anti-CD200L antibody and detecting binding between CD200L andthe antibody.

In another aspect, a method of diagnosing endometriosis in a subject isprovided. The method comprises: a. obtaining a secretory phaseendometrial biological sample from the subject, b. determining the levelof CD200L in the biological sample, c. comparing the level of CD200L inthe biological sample with the level of CD200L in a comparator, whereinwhen the level of CD200L in the biological sample is higher than thelevel of CD200L in the comparator, the subject is diagnosed withendometriosis. In an embodiment, the method includes the step oftreating the subject.

In another aspect, a method of monitoring a mammal following treatmentof endometriosis is provided. The method comprises: determining thelevel of CD200L in a biological sample from the mammal and comparing theCD200L level to a control pre-treatment CD200L control level todetermine if the CD200L level is reduced compared to the control level;and determining that the mammal is responding to treatment when theCD200L level is reduced in comparison to the control level.

In another aspect, a method of screening for a therapeutic agent usefulfor treating endometriosis is provided. The method comprises: contactingCD200L, a CD200R or both CD200L and a CD200R with the target agent; anddetecting whether or not the agent binds CD200L or the CD200R, orotherwise modulates the function or expression of CD200L or the CD200R,wherein detection of binding to, or modulation of the function orexpression of, CD200L or CD200R, indicates that the agent is a potentialtherapeutic agent that may be useful to treat endometriosis.

In another aspect, a method of treating endometriosis is provided. Themethod comprises; administering a therapeutically effective amount of acompound that inhibits CD200L or a CD200R to a subject in need oftreatment.

In a further aspect of the invention, a kit is provided comprising aCD200L-specific reactant for use in a method to detect the amount ofCD200L in a secretory phase endometrial biological sample.

These and other aspects will become apparent by reference to thefollowing Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are described in more depth by reference tothe following figures.

FIG. 1 is a depiction of the Endometriotic Deposits of 5 patientsstained for CD200 and CD200R (dark staining). Samples EM1p and EM2p wereperitoneal deposits (p) from patients in the proliferative phase oftheir menstrual cycle, and EM3p, EM4p, and EM5p were in the secretoryphase of their cycle. EM5ad shows an endometriotic deposit in thispatient's myometrium, called adenomyosis (ad). (100× magnification).c=cystic area, cc+chocolate cyst

FIG. 2 depicts the proliferative phase endometrium of patients withendometriotic deposits (EM) shown in FIG. 1 , compared to theproliferative phase endometrium from control women with no evidentendometriosis (NE) (100× magnification). e=luminal epithelium.

FIG. 3 depicts the secretory phase Endometrium of patients with.endometriosis (EM) compared to the secretory phase endometrium fromcontrol women with no evident endometriosis (NE) (100× magnification).

FIG. 4 is a High power (400× magnification) view of endometrium ofnormal NE patients 1, 2, 3, and 4, and endometriosis EM patients 1, 2,3, 4, 5 and corresponding peritoneal endometriotic deposits from each EMpatient. Arrows point to notably dense (dark) staining of CD200.

FIG. 5 shows low power 10× views of full thickness sections of uteriwith endometrial stripe (es) and myometrium (m).

FIG. 6 shows 400× photos of different types of stromal deposits (A, B,or C) in normal tissue (NE) and endometriosis tissue (EM) from theproliferative phase and secretory phase.

FIG. 7 shows Quantitative real-time polymerase chain reaction (qRT-PCR)values for proliferative, secretory, and menstrual phase endometrialbiopsy tissue from patients without endometriosis (NE) and withendometriosis (EM) are displayed on a log₂ scale. The data pointsrepresent fold increase with primers for CD200 compared to GAPDHcontrol. The geometric mean is provided in 2 columns comparing EM to NEAbove each display is the mean±1 sem value, and a P value based onStudent's t test (2 tail). For the left hand panel, the data representarithmetic means of the fold increase, whereas for the right hand panel,the mean log₂ value±1 sem and P value is provided.

FIG. 8 demonstrates qRT-PCR values as described in FIG. 7 legend, butusing primers for CD200R1.

FIG. 9 provides qRT-PCR values as described in FIG. 7 legend, butprimers for CD200R2 were used.

FIG. 10 graphically illustrates the results of a determination of sCD200in peripheral venous blood serum of patients with endometriosis vs.control samples using R&D Systems ELISA.

FIG. 11 illustrates the amino acid sequences of isoforms of cD200L(A/B/C) and the mRNA sequence of transcript 1 for CD200L (D).

FIG. 12 illustrates the amino acid (A) and mRNA (B) sequence of CD200R1.

FIG. 13 graphically illustrates levels of sCD200 in peripheral venousblood serum of patients with endometriosis (EM) vs. control (NE) samplesusing the Raybiotech ELISA for proliferative, secretory, and menstrualphase samples.

FIG. 14 provides a composite analysis using secretory phase data. Casespecific ratios of CD200R1 and R2 levels together with the sum ofCD200R1+CD200R2 for secretory phase patients are shown (A), and a piediagram comparing the relative percentage of CD200R1+CD200R2 that wasCD200R1 and CD200R2 comparing EM proliferative, secretory and menstrualcases to NE cases in the same menstrual cycle phase (B).

FIG. 15 provides (A) photomicrographs (200× magnification—scale bar 100nm) illustrating immunohistochemical staining of control (NE) andendometriosis (EM) secretory phase endometrium with CD200S antibody; (B)immunohistochemical staining of peritoneal endometrioma depositscorresponding to the EM cases in FIG. 5 ; and (C) immunohistochemicalstaining of endometriosis deposits found within the endometrium asadenomyosis.

DETAILED DESCRIPTION

A method of detecting CD200L in a secretory phase biological sample froma female mammal or subject is provided. The method comprises: i)obtaining the secretory phase biological sample from the subject, andii) detecting whether or not CD200L is present in the biological sampleby contacting the sample with an anti-CD200L antibody and detectingbinding between CD200L and the antibody.

The female mammal may be a human or non-human mammal, and may bepre-symptomatic for endometriosis or may be displaying clinical symptomssuggestive of endometriosis.

CD200 or Cluster of Differentiation 200, also referred to as OX-2membrane glycoprotein, is a type-1 membrane glycoprotein, contains twoimmunoglobulin domains, and thus belongs to the immunoglobulinsuperfamily. The term “CD200L” as used herein is meant to encompassmammalian CD200L, i.e. the wild-type non-truncated version incorporatingexon 2, including functional equivalents thereof, such as isoforms,variants and orthologs. The canonical amino acid sequence of CD200L,isoform 1, is UniProt P41217-1 (see FIG. 11A) having 278 AA and anintracellular tail (amino acids 268-278). Isoform 2 (P41217-2) has268-279 AA depending on whether the intracellular tail is as above or isjust EP. Isoform 3 (P41217-3) has 269 AA, includes a 25 amino acidleader sequence, and EP as the intracellular tail. Variants includethose in which one or more amino acids are altered without effect onfunction, e.g. amino acid at position 11 is C instead of S; amino acidat position 46 if T instead of P; and amino acid at position 76 is Ginstead of V. An example of a functionally equivalent ortholog includesa mouse protein encoded by a transcript such as NM_010818 (NCBI).

The present method includes the step of obtaining a secretory ormenstrual phase endometrial biological sample. A secretory or menstrualphase biological sample includes a sample originating from theendometrium of the subject during the secretory and/or menstrual phaseof the uterus. The sample may include peripheral blood, serum or plasma,urine, menstrual blood and/or discharge, uterine fluid, peritonealfluid, an endometrial tissue sample (biopsy), and lymphatic or vascularfluid from secretory phase endometrium. The sample may be obtained usingmethods known in the art. Preferred samples include those obtained bynon-invasive methods, such as shed endometrial fragments with bloodpresent in menstrual discharge, preferably collected using a menstrualcup but also extractable from tampons. The amount of sample requiredwill vary with the technique used to detect CD200L within the sample.Using an immunoassay such as an enzyme-linked immunoassay, an amount inthe range of 0.1-0.3 ml of cell-free fluid may be sufficient. For tissuebiopsy samples, generally 3-5 slides may be required.

Once the sample is obtained, it is then determined whether or notfull-length CD200 (CD200L) is present in the sample. The presence ofCD200L in the sample may be determined using a method that candistinguish between CD200L and the truncated form of CD200 (CD200tr orCD200S), a form resulting from a transcript missing exon 2, and thus,detect only CD200L.

In one embodiment, an immunoassay may be utilized to detect CD200L inthe biological sample in which the sample is contacted with ananti-CD200L antibody to permit detection of CD200L. Suitable antibodiesinclude those that detect the full-length CD200L and do not detect thetruncated version (CD200tr or CD200S), i.e. antibodies that target anantigen within an amino acid region of CD200L encoded by exon 2 (e.g. anantibody raised against amino acids 45-95 of CD200L). Examples ofsuitable antibodies include, but are not limited to, a polyclonalantibody from Bioss (catalogue #bs-6030R). It may be advantageous todetermine in a sample the percentage of CD200L and of CD200S. This isachieved by detecting CD200L as above using an antibody raised againstamino acids encoded by exon 2, and detecting CD200L +CD200S using anantibody raised against amino acids encoded by exon 3 (i.e. amino acids170-220, in the part of exon 3 not lost by the truncation of CD200L toform CD200S, namely a region beginning at amino acid position 124) suchas a polyclonal antibody from Bioss (ABIN3187966). The results may bevalidated by Western blots of cell lysates that contain mRNA for CD200Land CD200S, using the primer sequences such as those published byKobayashi et al. 2016).

Suitable immunoassays that may be employed to detect CD200L include, butare not limited to, enzyme-linked immunosorbent assays (ELISAs) orenzyme immunoassays (EIAs) in which an enzyme such as horseradishperoxidase (HRP), alkaline phosphatase (AP) or glucose oxidase permitsdetection of CD200L by emitting a detectable change (e.g. light, colourof chemiluminescence) in the presence of a certain reagent. Aradioimmunoassay (RIA) may also be used to detect CD200L in whichradioactive isotopes (e.g. ³²P, ¹²⁵I, ¹⁴C and ³H) bound to theantibody-antigen complex emits radioactivity that may be detected.Immunoassays which use fluorogenic reporters such as phycoerythrin,fluorescein, rhodamine or cyanine dye, or which useelectrochemiluminescent labels, such as ruthenium or terbium labels, maybe used to detect CD200L.

Polymerase chain reaction (PCR) may also be used to detect CD200L in abiological sample utilizing nucleotide primer pairs that target exon 2of CD200L DNA. In a preferred embodiment, Real-Time PCR or quantitativePCR (qPCR) may be used in which nucleic acid amplification and detectionis conducted in a single step utilizing fluorescent labelled probes thathybridize to the target CD200L sequence and fluoresce when displacedduring primer extension. As will be appreciated by one of skill in theart, suitable primer sets and probes for use in the present method aredesigned to hybridize to exon 2 of CD200L. Primer sequences aregenerally about 15 to 40 nucleotides in length, preferably 18-30nucleotides in length, and are complementary to the terminal ends of atarget exon 2 sequence. A probe, on the other hand, may be larger (e.g.100 or more nucleotides) and is designed to hybridize to a region thatis between the primer regions. As one of skill in the art willappreciate, primers and probes specific for exon 2 of CD200L may bedesigned based on the known sequence of CD200L. Software may be usedwhich has been developed for this purpose. Examples of primers includethose which target exon 2 (e.g. nucleotides nucleotides 361 . . . 687 ofCD200L-encoding nucleic acid). The primers may span exons 2 and 3 andbegin in exon 2 at a site that encodes amino acid at position 36 of theCD200L.

Detection of the level or concentration of CD200L in a secretory phaseendometrial biological sample is useful in a method of diagnosingendometriosis in a subject. The level of CD200L in the biological sampleis compared with the level of CD200L in a comparator, e.g. a controllevel of CD200L which reflects the level of CD200L in a correspondingsecretory phase endometrial biological sample from a healthy femalesubject or population, i.e. females that do not have endometriosis. Thecomparator may also be a wild-type control level, a historical controllevel and a historical norm. Detection of a level of CD200L in thebiological sample that is higher than the level of CD200L of thecomparator non-endometriosis patients that show that no numericaloverlap, e.g. such as 1 or 2 times higher, about 5 times higher or more,such as 8-10 times higher, 15 times higher, 20 times higher or more,indicates that subject is at risk of or has endometriosis. The level ofCD200L may also be compared to CD200S in the sample as described above.When the ratio of CD200L/CD200S is high (e.g. level CD200L is greaterthan CD200S, evidenced by an increase in endometrial deposits sinceCD200L appears to be associated with endometrial deposits), this isindicative of endometriosis or risk of in a subject. A ratio ofCD200L/CD200S that is decreased (e.g. level of CD200S is greater thanCD200L, evidenced by rejection of endometrial deposits when tissueenters the peritoneal cavity at the time of menstruation) there is areduced risk of endometriosis. Thus, CD200L is believed to possessimmunosuppressive properties that permit engraftment of endometrialcells on peritoneal surface, while CD200S is believed to antagonize theimmunosuppressive properties of CD200L.

The present method may also include the step of treating a subjectdetermined to have endometriosis. Suitable treatments includeadministration of pain medication including NSAIDs (nonsteroidalanti-inflammatory drugs) such as ibuprofen or naproxen, or opioids suchas codeine, fentanyl or methadone; hormone treatment that slowsendometrial tissue growth and prevent new implants of endometrial tissueincluding administration of contraceptives including estrogen andprogestin, or progestin alone; administration of gonadotropin-releasinghormone (GnRH) agonists such as buserelin, histrelin, leuprorelin,triptorelin, letrozole goserelin. These treatments are administered in atherapeutically effective amount, i.e. an amount suitable to treat orreduce the symptoms associated with endometriosis while not causingunacceptable adverse effects.

In another embodiment, endometriosis may be treated by administration ofa therapeutically effective amount of a compound that inhibits theinteraction of CD200L with its receptor, a CD200 receptor (CD200R suchas CD200R1). The compound is not particularly restricted and may be apeptide, protein, peptidomimetic, antibody or antigenic fragment thereofthat targets either CD200L or CD200R1, CD200L or CD200R1 bindingfragment, soluble CD200R polypeptide, nucleotide such as an anti-sensenucleic acid or siRNA that targets either CD200L or CD200R1-encodingnucleic acid, or a small molecule inhibitory compound.

Examples of suitable inhibitors include antibodies such as, but notlimited to, samalizumab, rituximab, ofatumumab, TRU-015, veltuzumab,ocrelizumab, or AME-133v. Other inhibitors of the CD200L/CD200Rinteraction include peptide inhibitors such as peptides derived fromCD200L and CD200R1, including peptides from the N-terminal region ofthese proteins, for example, V-domain peptides from the CDR2 and CDR3regions (e.g. a CD200L peptide comprising amino acids 62-75 and apeptide comprising amino acids 132-147), as well as 5-20 amino acidpeptides as described in WO2017079335 and peptidomimetics derived therefrom. DNA-based inhibitors may also used, including antisense and siRNAmolecules which are based on the mRNA or gene sequence of CD200, thatbind to CD200 and prevent the CD200L/CD200R interaction. DNA aptamersthat selectively recognize CD200R1 such as those described in US99385330may also be used. Other CD200 inhibitors have been described byKretz-Rommel (Journal of Immunology, 2008, 699-705); Chen (InternationalImmunology, 17(3), 289-296 (2005)); Gorczynski (International ScholarlyResearch Network, ISRN Immunology, Volume 2012, Article ID 682168; pages1-18); and in US Patent Publication No. 2002/0168364; U.S. Pat. No.6,955,811; or U.S. Pat. No. 7,902,151, that may be used. The relevantportions of these references are incorporated herein by reference.

To complement this therapeutic approach, or as a stand-alone approach,CD200S may also be targeted to inhibit the initial inflammatory responserequired for implantation of ectopic tissue. As one of skill in the artwill appreciate, the compound is not particularly restricted and may bea peptide, protein, peptidomimetic, antibody or antigenic fragmentthereof, or small molecule inhibitory compound, that targets eitherCD200S or its receptor, or a nucleotide such as an anti-sense nucleicacid or siRNA that targets either the CD200S gene, or nucleic acidencoding its receptor.

In another aspect of the invention, a method to identify a targettherapeutic agent useful for treating endometriosis is provided. Themethod comprises the steps of: contacting CD200L, a CD200R (such asCD200R1) or both CD200L and a CD200R with the target agent; anddetecting whether or not the agent binds CD200L or the CD200R, whereinif the agent binds CD200L or CD200R, then the agent is a potentialtherapeutic agent that may be useful to treat endometriosis. The methodmay include the additional step of identifying whether or not the agentinhibits the interaction between CD200L and a CD200R to further confirmits potential use as a treatment for endometriosis.

In another aspect of the present invention, a method of monitoring afemale mammal following treatment of endometriosis is provided. Themethod comprises: detecting the level of CD200L in a secretory phaseendometrial biological sample from the mammal; comparing the detectedCD200L level to a healthy control level or to a pre-treatment CD200Llevel in the mammal; and identifying that the mammal is responding totreatment when the CD200L level is reduced in comparison to thepre-treatment CD200L level, and/or is approaching the CD200L of thehealthy control level.

In yet another aspect, a method of identifying risk of infertility in afemale mammal is provided. The method comprises detecting the level ofCD200R1 and CD200R2 in a biological sample from the mammal, comparingthe level of CD200R1 to CD200R2 in the biological sample, andidentifying that the mammal is at risk of infertility when the level ofCD200R2 is higher than the level of CD200R1 in the biological sample.The biological sample may be a proliferative, secretory or menstrualphase sample. Preferably, the sample is a secretory phase sample.

In a further aspect of the invention, a kit is provided comprising aCD200L-specific reactant for use in a method to detect the amount ofCD200L in a secretory phase endometrial biological sample. Examples ofCD200L-specific reactants include antibodies that target exon 2 ofCD200L as exemplified above. The kit may additionally include materialsrequired to obtain a suitable sample such as means to collect menstrualblood. The kit may also optionally include instructions for use in themethod, and/or instructions with respect to the diagnosis ofendometriosis based on the detected amount of CD200L in the biologicalsample as detailed above.

Definitions

Unless otherwise indicated, the definitions and embodiments described inthis and other sections are intended to be applicable to all embodimentsand aspects of the present application herein described for which theyare suitable as would be understood by a person skilled in the art.

In understanding the scope of the present application, the term“comprising” and its derivatives, as used herein, are intended to beopen ended terms that specify the presence of the stated features,elements, components, groups, integers, and/or steps, but do not excludethe presence of other unstated features, elements, components, groups,integers and/or steps. The foregoing also applies to words havingsimilar meanings such as the terms, “including”, “having” and theirderivatives. The term “consisting” and its derivatives, as used herein,are intended to be closed terms that specify the presence of the statedfeatures, elements, components, groups, integers, and/or steps, butexclude the presence of other unstated features, elements, components,groups, integers and/or steps. The term “consisting essentially of”, asused herein, is intended to specify the presence of the stated features,elements, components, groups, integers, and/or steps as well as thosethat do not materially affect the basic and novel characteristic(s) offeatures, elements, components, groups, integers, and/or steps.

Terms of degree such as “substantially”, “about” and “approximately” asused herein mean a reasonable amount of deviation of the modified termsuch that the end result is not significantly changed. These terms ofdegree should be construed as including a deviation of at least ±5% ofthe modified term if this deviation would not negate the meaning of theword it modifies.

As used in this application, the singular forms “a”, “an” and “the”include plural references unless the content clearly dictates otherwise.For example, an embodiment including “an antibody” should be understoodto present certain aspects with one substance or two or more additionalsubstances.

In embodiments comprising an “additional” or “second” component, such asan additional or second antibody, the second component as used herein ischemically different from the other components or first component. A“third” component is different from the other, first, and secondcomponents, and further enumerated or “additional” components aresimilarly different.

The term “and/or” as used herein means that the listed items arepresent, or used, individually or in combination. In effect, this termmeans that “at least one of” or “one or more” of the listed items isused or present.

EXAMPLES Example 1

The following examples illustrate the scope of the invention. Specificelements of the example are for descriptive purposes only and are notintended to limit the scope of the invention. Those skilled in the artcould develop equivalent methods and utilize comparable materials thatare within the scope of the invention.

EXPERIMENTAL METHODS AND PROCEDURES

Patient materials: Archived normal pre-menopausal uteri from women ormatched hysterectomy specimens and simultaneously resected peritonealendometriotic implants were used. Hysterectomy specimens from womenwithout endometriosis were used as controls. Table 1 summarizes patientdetails. The study was approved by the Hamilton Integrated ResearchEthics Board.

TABLE 1 Properties of patients that were studied usingimmunohistochemistry Designation Age Symptoms requiring hysterectomyCycle phase Adenomyosis EM1 37 pelvic pain + dyspareunia ProliferativeNo EM2 41 pelvic pain + dyspareunia + Proliferative No fibroids EM3 41pelvic pain + dyspareunia Secretory No EM4 40 pelvic pain Secretory NoEM5 47 pelvic pain + menorrhagia + Secretory Yes fibroids + anteriorserosal adhesions NE1 45 uterine prolapsed with incontinence SecretoryNo NE2 41 menorrhagia Secretory No NE3 32 dysmenorrhea + small fibroidProliferative No NE4 40 symptomatic fibroids Proliferative No

An additional cohort of women diagnosed with laparoscopically-provenendometriosis (EM) and a control group (NE) composed of women withoutevidence of endometriosis (but pelvic pain) were recruited and consentedto providing endometrial biopsy tissue and a peripheral blood plasma andserum sample. The plasma samples were used in a previous study showingincreased brain-derived neurotrophic factor (BDNF) in endometriosis(Wessels et al., 2016). The mean (±standard deviation) age for womenwith endometriosis (34.5±6.71) did not differ from women in the controlgroup (35.4±8.29). All study participants reported regular menstrualcycles and current medication use reported by study participantsexcluded ovarian suppressing medications. All participants wereself-reported non-smokers.

Antibodies and Immunohistochemistry (IHC) staining: The method isdescribed in detail in Clark et al. (2017). Briefly, immunostaining wasperformed in the Department of Pathology's Immunohistology Facility inthe Michael DeGroote Centre for Learning and Discovery at McMasterUniversity using 4μ sections cut from paraffin blocks affixed topositively-charged slides. In the initial set of IHC studies antigenrecovery was done by heating in EDTA buffer pH8 (for anti-CD200 andanti-CD200R) or citrate buffer pH 6 (for Ki67) in a Biacore DigitalDecloaking Chamber using factory settings. Rabbit anti-human CD200 (RB846) serum and pre-immune control rabbit serum a kind gift from Dr. R.Gorczynski and described in detail elsewhere was used at a 1/1000dilution (Clark et al., 2017). In particular, immunohistochemicalstaining for human CD200 employed a rabbit polyclonal antibody raisedagainst the extracellular V+C regions of CD200 that were geneticallyattached to the gene sequence coding the AA of the Fc fragment of IgG inorder to render the CD200 soluble. Rabbit anti-Fc activity was absorbedout using an anti-Fc column. It is believed that this antibody reactswith AA sequences encoded by exons 1, 2, and 3. We also used ananti-CD200R raised against a 150-200 AA fragment of CD200R2(Uniprot/UniParc accession ID Q6Q8B3-1) coupled to KLH andantigen-affinity purified (Antibodies-online ABIN1715098,anti-CD200R1L). According to the published Antibodies-onlinespecifications, in Western blots this antibody detected a strong 37 kDand a weaker 30 kD band compatible with classical CD200R1 (325 AA) andCD200R1L (CD200R2) (271 AA) absent glycosylation respectively. There area number of shared amino acid (AA) sequence regions, one as long as 46AA, between CD200R1 and CD200R1L (CD200R2) that would make it likely fora polyclonal antiserum to react with both receptors (Wright et al.,2003). For this reason, we have used the CD200R designation throughoutthis document except where classical CD200R1/R1L are indicated.

In the second set of studies, the slides were stained for CD200L usingrabbit antigen-affinity purified antibody for AA 45-95 of the conicalsequence of CD200 canonical isoform a, (Uniprot/UniParc accession IDP41217-1) (Antibodies Online ABIN761396) and for CD200S using rabbitanti-human CD200 antibodies generated against the amino acid (AA)170-220 sequence of the canonical isoform a, P41217-1(Antibodies-online, ABIN318966). Rabbit anti-KLH antibody(Antibodies-online, ABIN401183) was used as a control. All antibodieswere diluted 1:200 in Power Vision IHC Super Blocker (Leica) prior tostaining. Slides were stained using the Bond Polymer Refine Detectionkit (Leica). Stained slides were digitally scanned and analyzed usingImagescope (Leica), and photographed at 200× and 400× . Digital colourphotomicrographs at 400× were taken using Imagescope from 11-13different areas/specimen beginning at the epithelium and extending toglandular regions in the luminal half of the endometrium. Each CD200S⁺cell (nucleus+intracellular staining) was numbered so that no cell wasmissed and no cell was double counted. Using the scale bar of 50 μm, thearea represented 0.0335 mm², and given a uniform 4 μm thickness of eachsection, a unit volume of 1340 μm³ or 0.000134 mm³. The CD200S+cellcount reflects cell number per unit volume. The number of positive cellsin 11-13 photomicrographs was used to calculate the mean and sem numberof cells per unit volume for each secretory phase endometriosis andnon-endometriosis cases.

The slides were scanned using Imagescope and photographed at 10× , 40×,100× and 400× into jpeg files. Image analysis was done using NIH Image J1.4 software. Briefly, using Image J software, 5 to 6 semi-rectangularareas of the endometrium at 10× magnification (or foci of specificinterest at higher power magnification) were outlined using the drawingtool (which allows precise inclusion of irregularly shaped borders) soas to provide intensity data in pixels from which a mean and standarderror of the mean could be calculated. The blue intensity with thecontrol antibody was subtracted from the red-brown+blue intensity ineach semi-rectangle with the corresponding antigen-specific stain.

qRT-PCR analysis: Samples of eutopic endometrium were obtained bypipelle biopsy during laparoscopic surgery for chronic pelvic pain andsuspicion of endometriosis. Study participants were assigned to control(no endometriosis) or cases (endometriosis) based on evidence ofendometriotic lesions noted by the surgeon in the operative report andconfirmation by histopathology in the pathology report. Samples weretransferred to the lab on ice where they were minced and suspended inRNAlater (SigmaAldrich Chemical Co. Oakville, ON) for 24 hr at 4° C. andthen transferred for storage 85° C. until required for analysis.

Sample RNA was nano-dropped and reverse transcribed into cDNA using theiScript cDNA Synthesis Kit (Bio-Rad). Each RT reaction consisted of 4 ulof 5× iScript reaction mix, 1 ul of iScript reverse transcriptase, andvariable volumes of Nuclease-free water and RNA template to produce acDNA volume of 200 ng. Each reaction was heated to 25° C. for 5 minutes,42° C. for 30 minutes and finally 85° C. for 5 minutes. Real-time qPCRwas completed using the CFX96 Touch Real-Time PCR Detection System(BioRad). Each sample was analyzed in triplicate on a 384-well plate.Each well contained a reaction mixture including 2.5 uL of sampletemplate cDNA, 1.5 uL RNAse-free water, 0.5 uL of the target Forward andReverse Primer** and 5 uL of SYBR Green. Each 384-well plate was heatedto 95° C. for 15 min, followed by 45 cycles at 94° C. for 10 seconds, 60° C. for 10 seconds and 72° C. for 10 seconds. This was followed by onecycle for melting curve acquisition.

ELISA analysis: An initial set of studies was conducted using R&DSystems' ELISA assay (DuoSet, Catalogue #D42724) according to themanufacturer's specifications. Ninety-six well ELISA plates were coatedwith their proprietary mouse monoclonal anti-human CD200 captureantibody, washed, and incubated with serial dilutions of the sCD200:Fccontrol antigen or with test peripheral blood serum fromnon-endometriosis controls (NE) or endometriosis patients (EM) duringthe menstrual phase. The sera were tested at ½, ¼. ⅛/⅙, and 1/32dilutions to be sure the result was not affected by inhibitors in theserum. Inhibitors in serum may include, but are not limited to, solubleCD200R1 to which CD200L binds, IgG to which sCD200 may bind, CD200Swhich may bind to the capture antibody and impede binding of CD200L. Asecond set of studies (see Example 2) was done using serum samples fromendometriosis patients EM (N=13) and non-endometriosis patients (withpelvic pain) NE (N=10) cases using a more sensitive commerciallyavailable RayBioTech sandwich Human CD200 ELISA kit (ELH-CD200,RayBioTech, Norcross, GA). Each individual plate assessed sCD200concentrations of four to five EM and three to four NE during either thesecretory, menstrual, or proliferative phase. The sera were tested at ½,¼. ⅛/⅙, and 1/32 dilutions to be sure the result was not affected byinhibitors in the serum. Inhibitors in serum may include, but may not belimited to, soluble CD200R1 to which CD200L binds, IgG to which sCD200may bind. Duplicate at a 1 in 2 dilution gave optimal results (Wong etal., 2016). Stage in the menstrual cycle was determined by the timeelapsed from date of the last menstrual period to the date of samplecollection. The optical density was measured using a BioTek Utility ofSynergy H4 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT) ata wavelength of 450 nm. CD200 concentrations were determined from astandard curve using different concentrations of the CD200 standard(P41217.3) and the resulting optical density values (OD) were fitted toa second order polynomial y=ax²+bx+c model as described by Herman et al.(2008) using Microsoft Excel. Here y=OD and x=soluble CD200concentration (including zero values). The concentration of sCD200 inindividual test wells based on the OD value was determined from theformula sCD200 pg/ml=(−b+SQRT(b²−4a(c−OD)))/(2a).

Statistics: The data was initially analyzed using Prism 8 (GraphPadSoftware, San Diego, CA, USA) and included tests for normality andlognormal distribution. In all cases except one where n was sufficient,a lognormal distribution was confirmed, and the data are shown infigures on a log scale along with the geometric mean. The mean andstandard error of the mean (SEM) for In transformed data was used tocalculate p using the parametric Student't test. When neither a normalnor lognormal data distribution occurred, the Fisher's Exact test wasused. In all comparisons, p values were confirmed using thenon-parametric Wilcoxon rank sum test and precise p values estimatedusing the online calculator of Navendu Vasavata 2016 (astatsa.com).Because the initial IHC study of CD200 expression in endometriosisindicated increased production of CD200 in the secretory phase and notproliferative phase of the menstrual cycle, with no change in stainingfor CD200R (which included CD200R1 and CD200R2) this a priori knowledgeeliminated the need for a Bonferonni correction that would have requiredfor multiple tests (N) of significance a p value≤0.05/N (at the risk offalse negatives, a type II error). In all cases, p≤0.05 was consideredto indicate statistical significance.

RESULTS CD200 and CD200R are Detected by Immunohistochemistry inPeritoneal Endometriosis Deposits in Both Proliferative and SecretoryPhase of the Menstrual Cycle.

FIG. 1 shows staining for CD200 and CD200R in peritoneal endometriosisdeposits at 100× magnification. Dark foci of CD200⁺ and CD200R1⁺deposits in endometrial epithelium forming cyst walls and in the stromawere evident in stroma, CD200⁺ and CD200R⁺ acellular material withincysts was compatible with shed/soluble membrane. A deposit in myometriumin EM5 (adenomyosis) was also positive for both markers. Note lack ofstaining of the myometrium. There was no striking difference betweenstaining of ectopic deposits in the proliferative phase of the menstrualcycle compared to the secretory phase. These data raised the question,as to whether or not uterine endometrium from these same cases showedsimilar staining, and if so, was it different from staining in theendometrium of patients with no evidence of endometriosis (NE).

CD200 and CD200R Staining is Present in Proliferative Phase EndometriumFrom Both Non-Endometriosis Controls (NE) and Endometriosis Patients(EM)

FIG. 2 shows that CD200 and CD200R staining was present in proliferativephase endometrium from both 2 non-endometriosis controls (NE) and 2endometriosis patients (EM) at 100× magnification. The luminalepithelium stained as well as the epithelium of endometrial glands whichin some sections can be seen communicating with the uterine lumen. Therewas also scattered dark spot staining in stroma which is not as welldefined as in epithelium. Note the pre-immune and anti-KLH controlantibody as well as diluent control (no antibody used) provides thecomparator defining absent staining.

CD200 and CD200R Staining is Present in Secretory Phase Endometrium FromBoth Non-Endometriosis Controls (NE) and Endometriosis Patients (EM)

FIG. 3 shows 100× magnification photomicrographs similar to FIG. 2 ofendometrium in uterine sections done in the secretory phase of themenstrual cycle from 2 control patients who did not have endometriosisand 3 with endometriosis. Again the epithelium of glands is stained forCD200 and CD200R. More striking staining for CD200 and CD200R was notedin EM4 and EM5 which appear to be late in the secretory phase assuperficial endometrium is beginning to slough. At this magnification,details of variable staining in stroma is less clear than in epithelium.Staining of CD200 and CD200R is also present in acellular materialwithin the lumens of endometrial glands.

CD200 and CD200R Increased in Luteal Phase Endometrium of Patients WithEndometriosis

The 100× magnification images in FIGS. 1-3 provide a general picture ofstaining for CD200 and CD200R, but as already pointed out, detailedinformation about staining in stroma was minimal. Therefore 400×photomicrographs were examined.

FIG. 4 shows the result with 4 control patients and 5 endometriosispatients including the proliferative and secretory phases of the cycle.It can be seen that both glandular epithelium and some cells in thestroma stained for both proteins, but staining appeared to be increasedin the luteal phase endometrium of patients with endometriosisparticularly in the epithelial of endometrium about to be shed bymenstruation. CD200⁺ and CD200R⁺ acellular material was frequentlypresent in endometrial gland lumens. Staining is quantified in Table 2.FIG. 4 left set of 2 panels shows CD200 staining of glandular epitheliumof NE cases and staining of channels in stroma that appear to be venulescontaining erythrocytes or lymphatics where erythrocytes were not seen:in ¾ cases, staining for CD200R was undetectable. In the middle set of 2panels from EM cases, a similar result for CD200 staining was noted in5/5 cases, and in only ⅕ cases (#4) was there weak staining for CD200R.In the right set of 2 panels, 5/5 endometriotic deposits showed densestaining of the contents of venules with possibly weak staining in thelumen in ⅕ (#5) and weak venule wall staining in ⅕ (#3). Based onstaining intensity, CD200 was>>> and CD200R staining.

TABLE 2 Quantitative analysis of expression of CD200 and CD200R1L infull thickness endometrium of normal (NE) and endometriosis (EM) womenProliferative Phase Secretory Phase CD200^(a) CD200R CD200 CD200R NE317.9 ± 1.2 (5)^(ab) 38.9 ± 2.8 (5) NE1 11.8 ± 2.5 (5) 19.8 ± 1.0 (5) NE48.5 ± 1.0 (5) 11.7 ± 0.5 (5) NE2 17.6 ± 1.7 (5) 30.6 ± 1.5 (5) μ^(c) =13.2 ± 1.5 25.3 ± 2.8 14.7 ± 3.0 25.2 ± 1.8 EM1 9.4 ± 0.5 (5) 12.7 ± 1.6(5) EM3 16.9 ± 1.8 (5) 24.9 ± 1.8 (5) EM2 16.5 ± 2.4 (5) 20.8 ± 1.4 (5)EM4 23.9 ± 1.6 (5) 25.0 ± 1.3 (5) EM5 8.0 ± 1.9 (5) 20.6 ± 1.9 (5) μ^(c)= 13.0 ± 2.5 16.8 ± 2.1 16.3 ± 2.2^(d) 23.5 ± 2.1 ^(a)anti-CD200 1/1000intensity mean and 1 sem after subtraction of pre-immune control 1/1000^(b)parenthesis show number of areas measured. ^(c)mean ± 1 sem. ^(d)themean value of 16.3 ± 2.2 in the EM secretory phase group was greaterthan in the proliferative phase and in NE secretory and proliferativephases but this trend did not achieve statistical significance t = 1.01,1.16, 0.43 respectively.

Quantification of CD200 and CD200R in Endometrial Stripes of WholeUterus Images

FIG. 5 shows low power (10× magnification) whole uterus images. UsingImage J software, the endometrial stripe (stained with anti-CD200) wasdivided into 5-6 adjacent semi-rectangular areas and red versus bluestaining intensity was measured. In the black and white version of thefigures, red is dark and blue is white. The result for full thicknessendometrium is shown in Table 2. The data for CD200 represents thenumber of pixels with anti-CD200 minus the number of pixels withpre-immune control serum, and for CD200R, the number of pixels withanti-CD200R minus the number of pixels with control anti-KLH. The meanand 1 standard error of the mean (sem) was determined using the 5-6semi-rectangular areas that encompassed the entire endometrial stripe.The mean and 1 sem for each group of 2 or 3 patients was thencalculated. There was more anti-CD200R staining than anti-CD200, and aslight trend to more CD200 in the secretory phase, although the latterwas more evident in the proliferative phase EM group, due to lowerCD200R staining. Due to the small sample size, none of the differencesachieved statistical significance.

Analysis of Luminal Portion of Endometrium

As it is the luminal component between the basal layer that abutsmyometrium and the luminal surface that will be shed and pass in aretrograde manner into the peritoneal cavity, that component of theendometrium is potentially of greater importance, and so a similaranalysis of the luminal ½ of the endometrium abutting the lumen was doneand is shown in Table 3. As shown, the result is similar to the outcomein FIG. 2 except for a more prominent increase in CD200 in EM secretoryphase. Indeed, in FIG. 3 , in late phase endometrium the stainingintensity was striking. However, due to small sample size, the increasein CD200 staining did not achieve statistical significance.

TABLE 3 Quantitative analysis of expression of CD200 and CD200R in inner(luminal) half of endometrium of normal (NE) and endometriosis (EM)women Proliferative Phase Secretory Phase CD200 CD200R CD200 CD200R NE319.8 ± 2.2 (5) 47.8 ± 4.6 (5) NE1 10.5 ± 3.2 (5) 20.0 ± 1.2 (5) NE4 8.9± 1.6 (5) 12.9 ± 0.6 (5) NE2 21.3 ± 1.5 (5) 35.0 ± 1.2 (5) μ = 14.4 ±2.7 30.4 ± 4.6 15.7 ± 1.7 27.5 ± 1.7 EM1 10.4 ± 1.3 (5) 11.2 ± 1.4 (5)EM3 18.0 ± 2.3 (5) 28.5 ± 2.7 (5) EM2 16.6 ± 4.8 (6) 25.5 ± 2.4 (6) EM427.2 ± 2.6 (5) 28.4 ± 1.5 (5) EM5 11.3 ± 1.6 (5) 20.6 ± 1.5 (5) μ^(a) =13.5 ± 5.0 18.4 ± 2.8 18.8 ± 2.6^(b) 25.8 ± 2.4 ^(a)Mean and 1 sem.^(b,c)The mean CD200 staining of 18.8 ± 2.6 from 3 EM secretory phasecases versus 13.5 in EM proliferative phase and 15.7 ± 1.7 NE secretoryand 14.4 ± 2.7 NE proliferative phase groups achieved t = 0.94, 1.08,1.47 respectively.

Quantification of Staining in Walls of Venules and Lymphatic Channels inEndometrial Stroma

To further investigate the staining of non-epithelial stromal venulesand lymphatic channels noted in FIG. 4 , Image J analysis was done.Venules were identifiable by the presence of intra-luminal erythrocytes.It was difficult to find identical areas in the negative isotypecontrol-stained sections to compare to the stained tissues, but ascontrol antibody staining intensities are usually quite similar foranti-CD200 and anti-CD200R, and anti-CD200R staining of venule/lymphaticwalls was similar to adjacent stroma, the value of CD200-CD200R stainingintensity (Δ) was determined. For 4 normal patient endometrial, Δ was51.2±7.1 (8 measurements) and for 5 endometriosis patients, Δ wasslightly greater at 59.5+8.3 (10 measurements), which was notstatistically significant. In endometrium, unlike in endometriomas,there was no CD200 staining within the vascular lumens.

Expression of both CD200 and CD200R were detected in endometrioticdeposits and in a deposit of adenomyosis. In peritoneal deposits, smallareas compatible with venules and lymphatic channels that were intenselypositive for CD200 lacked CD200R expression, but as epithelial cystsformed, CD200R was detected in the epithelium and was usually>CD200staining intensity. Further, both CD200 and CD200R were detected in theproliferative and secretory phase endometrium of these patients. In fullthickness endometrium, Table 2 shows similar values for CD200 and CD200Rcomparing endometriosis patients to controls. CD200 was lower in theproliferative phase (16.8±2.1) but this was not statistically differentfrom normal control (25.3±2.8) (t=2.41, P<0.1 but in the secretoryphase, EM patient, CD200 staining intensity was greater than in theproliferative phase. However, the difference was not statisticallysignificant. In NE patients, CD200 did not increase in the secretoryphase, and was similar to the mean EM proliferative phase value.

Table 4 shows the result of analysis of endometrial stromal depositslarge enough for Image J quantitation according to the classificationshown in FIG. 6 . The Image J signal with anti-CD200 had the backgroundstaining with pre-immune rabbit serum subtracted. While in the secretoryphase, the mean EM staining was greater than in NE, the difference wasnot statistically significant. On further analysis, stromal depositscould be subdivided as shown in FIG. 6 . Type A deposits were linearcellular walls with no evident lumen, whereas B showed a cellular walland lumen containing erythrocytes or neutrophils and sometimes somefluid phase staining. Type C deposits were larger accumulations with aCD200⁺ fluid phase±wall staining, and sometimes erythrocytes werepresent (but were excluded in the Image J scoring). For purposes ofanalysis of conduits containing sCD200⁺ type B vessels where there wasobvious cell-free CD200⁺ fluid, these were denoted B-C and ultimatelygrouped with type C deposits.

It can be seen from the tabulation that the frequency of Type Caccumulations was significantly greater (P=0.000006, by Fisher's Exacttest) in the secretory phase EM tissues compared to secretory phase NEtissue, and in contrast to no evident difference comparing NE to EM inproliferative phase endometrium. Using Type C deposits, forproliferative endometrium the product of intensity×area×number ofdeposits per case averaged 2241±726 for NE versus 391±129 for EM (P+NSby Student's t test), whereas in secretory endometrium it was1748.9±187.1 for EM versus 186.7 in NE (no sem calculated as only 1value). The standard deviation of 1748.9 was 560.248 so that 186.7 wasP<0.00265 below the EM mean (z=1561.8/560.248 =2.787). The higher sCD200value in secretory phase EM vessels contrasted with the proliferativephase result where the NE value was>than the EM value. The data werecompatible with increased accumulation of sCD200 in thevascular/lymphatic lumens in secretory phase EM endometrium.

TABLE 4 Quantitative measurement of focal stromal CD200 stainingPROLIFERATIVE SECRETORY NE3 (19) NE4 (18) NE1 (20) NE2 (21) μ + sem56.89 ± 3.33 38.34 ± 3.01 59.57 ± 3.33 72.79 ± 4.31 EM1(20) EM2(11) EM3(41) EM4 (45) EM5 (27) μ ± sem 54.5 ± 3.15 76.0 ± 7.40 73.2 ± 3.51 74.0± 2.09 62.1 ± 4.30 PROLIFERATIVE SECRETORY VESSEL NE3 NE4 NE1 NE2 A 3 113 9 B 12 4 6 12 C 4 13 1 0 EM1 EM2 EM3 EM4 EM5 A 6 0 5 20 12 B 9 7 1317 5 C 5 4 22 8 10 A + B C A + B C NE3 + 4 20 17 NE1 + 2 40 1 EM1 + 2 229 EM3 + 4 + 5 72 40 Exact P = 0.12 Exact P = 0.000006

Collette et al. (2004) have reported elevation of endometrialproteolytic activity in normal and endometriosis patient endometrium,with a peak in the secretory phase, and although EM values fromindividual patients shows a 30% overlap of the NE range, increasedcellular expression of CD200 combined with increased proteolyticactivity can explain intense intralumenal staining of sCD200 invascular/lymphatic structures. Increased accumulation could also occurdue to reduced blood flow which has been documented in the endometriumof infertile patients (and EM causes infertility). Elevated sCD200 andCD200⁺ cells in menstrual blood would be a logical prediction.

CD200 mRNA Levels Significantly Increased in Secretory Phase Endometrium

To determine if the accumulation of sCD200 in the venules/lymphatics ofsecretory endometrium, particularly in the basal zone, was due toincreased generation of sCD200 by matrix metalloproteinases cleavingsurface CD200 (Wong et al., 2016; Collette et al., 2004) or due toincreased synthesis of CD200, qRT-PCR analysis of stored wholeendometrial samples was carried out. The primers used included:

CD200 F (SEQ ID NO: 1) 5′-acc cag gat gaa aga gag ca-3′ CD200 R(SEQ ID NO: 2) 5-tat agg cag gct gga tca cc-3′  CD200R1 F*(SEQ ID NO: 3) 5′-cca ttt gac tgg caa-3′ CD200R1 R* (SEQ ID NO: 4)5′-gca gcc att gac ttt caa ca-3′ CD200R2 F (SEQ ID NO: 5)5′-caa ggc agt tac agg gaa gc-3′ CD200R2 R (SEQ ID NO: 6)5′-gcc agt caa atg gga gac at-3′ *Designed at the University of Toronto(University Health Network) and is published in the public domain.

The amplification of CD200, CD200R1, and CD200R2 mRNA in the qRT-PCR wasexpressed as fold increase over GAPDH control provided by a QiagenQuantiTect Primer Assay (QT007924) which detects GAPDH transcriptsNM_0011256799, NM_002046, NM_001289745, and NM_001289746. The primersequences are proprietary, and include primers for GAPDH (QT007924) asfollows:

GAPDH F (SEQ ID NO: 7) 5′-tca acg acc act ttg tca aac ctc a-3′ GAPDH R(SEQ ID NO: 8) 5′-gct ggt ggt cca ggg gtc tta ct-3′

To determine if differences in serum sCD200 were related to differencesin CD200 production in secretory phase EM cases, mRNA for CD200,CD200R1, and CD200R2 was determined by RT-PCR. It can be seen thatcomparing CD200 mRNA levels in EM to NE cases in the proliferative phaseshowed no increase in EM, and that was true when the 2^(−ΔcT) valueswere also compared. In contrast, in secretory phase endometrium CD200mRNA levels were significantly increased in EM cases compared to NEcontrols. This was true also when −ΔCT values were considered (see FIG.7A/B).

As the null hypothesis was that CD200 levels would not be increased inEM, a 1 tail P value may be used so that P<0.025 was obtained. Theoverlap of EM with NE values in 2/6 cases could be related to stage ofthe secretory cycle from which these samples were taken or othertechnical factors. Even so, increased synthesis of CD200 was present inthe EM group compared to NE controls. A similar increase was not seenfor CD200R1 or CD200R2 mRNA levels (FIGS. 8 and 9 ) with this samplesize.

The detection of this increase which was not seen in theimmunohistochemical (IHC) quantitative analysis of CD200 staining inwhole endometrium. This may be for one or more of the following reasons.In preparing the blocks for IHC, during fixation, some soluble sCD200may have been lost. The polyclonal anti-CD200 antibody used for theinitial set of IHC studies was raised against the whole CD200:Fcmolecule, and anti-Fc activity was absorbed out. It is now known thatthere can be a truncated CD200 (called trCD200 or CD200S as distinctfrom CD200L). CD200S lacks exon 2 determinants of CD200 (Chen et al.,2006; Kobayashi et al., 2016). The primers used for qRT-PCR solelydetect CD200L mRNA, whereas the antibody used in the initial set of IHCstaining could potentially detect more epitopes of CD200L that theAntibodies Online anti-CD200L, and with the methodology used we cannotexclude the possibility that CD200S might also have been stained. AsCD200S stimulates rejection and blocks CD200L, this could result inreduced CD200L bioactivity. It is also noted that the qRT-PCR data inthe Figures shows that the amount of CD200R1 was greater than the amountof CD200R2 mRNA in the qRT-PCR analysis of NE endometrium, and insecretory phase EM endometrium, CD200R1 synthesis appeared to decreaseresulting in less CD200R1 able to bind to and neutralize CD200L. All ofthe CD200L epitopes that bind to CD200R1 are in exon 2 (Hatherley etal., 2005), and CD200S is missing exon 2, so which receptor CD200S bindsto in order to activate macrophages remains to be determined (Kobayashiet al., 2016).

ELISA Analysis of Peripheral Blood Serum From Secretory Phase NE and EMCases Demonstrates Increased sCD200 in the Secretory Phase of EMEndometrium.

The initial ELISA data clearly show elevated blood sCD200 in asignificant number of EM samples (see FIG. 10 ). The data can beconverted from OD into pg/ml using a standard curve based on apolynomial model. The R&D Systems ELISA kit exhibits sensitivity as ≥150pg/ml, and a more sensitive ELISA may have yielded improved results.While R&D Systems indicates their standard curve is linear between 150pg/ml and 10000 pg/ml, the results obtained did not fit a linear y=ax+bmodel. Therefore, in FIG. 10 , the OD 450 values are given. Thebackground from wells containing diluents alone and no sCD200:Fc orserum was subtracted from all measured OD levels. EM sera obtained frompatients in the menstrual phase of their cycle was shown to haveelevated levels of sCD200L. Data is shown from the ½ dilution, but a ¼dilution gave a similar result. In the 4 NE cases, the mean OD abovebackground was 0.0005125+0.000496 (1 sem). Any OD value above 0.00168exceeded 95% of predicted NE sample values above the mean. As can beseen, the elevated EM case values were above this threshold (1.5165,0.3260, 0.1476). Results using the more sensitive Raybiotech ELISA areprovided in Example 2 below.

Discussion

The presence of very intense deposits of CD200 in venules and veins inperitoneal endometriotic deposits was striking and different fromstaining of epithelium and non-vascular stromal elements. In thecorresponding endometrium there was strikingly positive staining ofCD200 in the walls of lymphatics and venules (identifiable by presenceof erythrocytes) which implies high levels of soluble CD200 (sCD200) atthe capillary level. sCD200 is cleaved from the cell surface and mayexert important biological effects at a distance by binding to CD200R⁺cells. Nevertheless, the difference in CD200 staining of the walls oflymphatic and venules in endometriosis patients' endometrium was onlyslightly greater than in normal non-endometriosis control endometrialsamples. From FIG. 4 , expression of epithelial CD200 at the time ofshedding may be quite high. CD200 derived from venular and lymphaticwalls is added to menstruated blood. On this basis, quantitativemeasurement of the concentration of CD200⁺ cells and sCD200 in menstrualblood provides an estimate of the dose of bioactive CD200 delivered tothe peritoneal cavity. That CD200 dose is much greater in EM patientsthan in NE patients and hence provides a reliable predictor fordiagnosing the presence of endometriosis and a predictor of thelikelihood of developing endometriosis.

A significant embodiment of the present data is the prediction that thelevel of sCD200 in menstrual blood and CD200 on menstrual blood cellsmay provide a more accurate predictor of endometriosis when standardizedto 17-β-estradiol levels that modulate CD200 mRNA levels. Differentembodiments of the invention have been shown by the above examples.Those skilled in the art could develop alternatives to the methodsmentioned above that are within the scope of the invention and definedclaims.

Example 2—ELISA Quantitation of sCD200 in Serum

Serum samples from endometriosis (EM) (N=13) and non-endometriosis (NE)(N=10) cases were assessed using a commercially available RayBioTechsandwich Human CD200 ELISA kit (ELH-CD200, RayBioTech, Norcross, GA)which could detect sCD200 levels as low as 20-30 pg/ml. As described inthe method of Example 1, after serial dilution results were examined, a½ dilution which others have also found optimal was used (Wong et al.,2016) given neat serum can show a prozone effect. Stage in the menstrualcycle was determined by the time elapsed from date of the last menstrualperiod to the date of sample collection. The optical density wasmeasured using a BioTek Utility of Synergy H4 Hybrid Multi-ModeMicroplate Reader (BioTek, Winooski, VT) at a wavelength of 450 nm.CD200 concentrations were determined from a standard curve usingdifferent concentrations of the CD200 standard (P41217.3) and theresulting optical density values (OD) were fitted to a second orderpolynomial y=ax²+bx+c model as described by Herman et al. (2008) usingMicrosoft Excel (2008). Here y=OD and x=soluble CD200 concentration(including zero values). The concentration of sCD200 in individual testwells based on the OD value was determined from the formula sCD200pg/ml=(−b+SQRT(b²−4a(c−OD)))/(2a).

Results:

The results of a determination of sCD200 in peripheral venous bloodserum of patients with endometriosis (EM) vs. control (NE) samples usingELISA for proliferative, secretory, and menstrual phases are shown inFIG. 13 . For secretory phase data, the EM values did not fit a normalor lognormal distribution and so the statistical test of the nullhypothesis that EM was not>NE was done using the non-parametric Wilcoxonrank sum test. The p value was 0.0083. As the EM and NE populations didnot overlap, classifying NE data<20 pg/ml as negative and EM data>20pg/ml as positive allowed use of Fisher's Exact test, an alternativenon-parametric statistic, and the p value was 0.0061. The proliferativeand menstrual EM and NE data did show a lognormal distribution, and themean In μ for 4 P NE cases was 3.75939±0.320341 compared to the ln μ for5 P EM cases 3.69586±0.53613, and Student's t test value of 0.1017 withp=0.461. The rank sum p value for the null hypothesis that EM was not>NEwas 0.500. For the menstrual cases, the mean ln μ for 3 NE cases was4.00543±0.61168 and for 5 EM cases, 3.16278±0.38541, Student's t testvalue of 1.170 with p=0.143. The rank sum p for null hypothesis that MEM was not>M NE was p=0.197. P, proliferative; S, secretory; M,menstrual; NE, no endometriosis controls; EM, endometriosis cases. Thus,increased secretory phase sCD200 serum levels is associated withendometriosis.

The data was compared to another potential marker for endometriosis,namely, BDNF, and the results are shown in Table 5. Secretory phaseplasma BDNF levels do not correlate with serum sCD200 levels. The resultconfirms serum CD200 to be a diagnostic marker of endometriosis.

TABLE 5 EM Case # BDNF value^(a) sCD200 pg/ml ± 1 sem^(b) No. 62 3,311.9POS 53.37 ± 16.68 POS No. 85 461.0 NEG 1513.90 ± 312.80 POS No. 177848.2 NEG 70.05 ± 10.01 POS No. 214 1,537.8 POS 60.04 ± 13.35 POS No.225 822.1 NEG 173.64 ± 0 POS No. 205 70.8 NEG 57.15 ± 33.05 POS^(c)^(a)data from Wessels et al. Fertil Steril 2016; 105: 119-28. BDNF > 999is positive ^(b)Based on duplicate wells (N = 2) at serum dilution of1:2; sCD200 > 0 was positive as all non-EM controls were ≤10 pg/ml.^(c)neat was 120.7 ± 17.7 pg/ml. This serum was tested in a separateELISA study where a neat serum sample was compared to a ½ dilution toassess prozone effects.

Example 3—CD200R1/CD200R2 Ratio in Secretory Phase EM Endometrial Tissue

Endometrial samples were retrieved via pipelle biopsy during surgery.Tissue samples were stabilized using RNAlater (Thermo Scientific,Mississauga, ON) and frozen until required for RNA analysis. Sampleswere weighed (30 mg) and placed into 700 μl of QIAzol Lysis Reagent(Qiagen, Toronto, ON) prior to homogenization (Bio-Gen, Oxford, CT).Total RNA was isolated from eutopic EM (n=17) and NE (n=8) endometrialsamples, using the miRNeasy mini kit (Qiagen). Primers for all genes(CD200, CD200R1, CD200R2) were designed using Primer BLAST softwareavailable at the NCBI website. qRT-PCR reactions were carried out usingforward and reverse primer sequences obtained from the McMasterUniversity molecular biology core facility (Mobix, McMaster University)and as described previously. RNA was nano-dropped (Thermo Scientific)and reverse transcribed into cDNA using the iScript cDNA Synthesis Kit(BioRad, Mississauga, ON). Each qRT-PCR reaction consisted of 4 μ1 of 5xiScript reaction mix, 1 μ1 of iScript reverse transcriptase, andvariable volumes of Nuclease-free water and RNA template to produce acDNA volume of 200 ng. Each reaction was sequentially heated to 25° C.for 5 minutes, 42° C. for 30 minutes and 85° C. for 5 minutes. Real-timeqPCR was performed using the CFX96 Touch Real-Time PCR Detection System(BioRad). Samples were analyzed in triplicate on a 384-well plate. Eachwell contained a reaction mixture including 2.5 μL of sample templatecDNA, 1.5 μL RNAse-free water, 0.5 μL of the target forward and reverseprimer and 5 uL of SYBR Green. Each 384-well plate was heated to 95° C.for 15 min, followed by 45 cycles at 94° C. for 10 seconds, 60° C. for10 seconds and 72° C. for 10 seconds. This was followed by one cycle formelting curve acquisition.

Results:

To determine if differences in serum sCD200 were related to differencesin CD200 production in secretory phase EM cases, mRNA for CD200,CD200R1, and CD200R2 was determined by RT-PCR in 24 eutopic EM and NEendometrial samples but only three samples also had serum sCD200measured, one secretory phase EM case, one proliferative phase NE, andone menstrual phase NE. In FIG. 14A, a total of eight EM and 3 NEsecretory phase cases are shown, and in this group, only one EM case hadhad serum CD200 measured. CD200 mRNA expression was significantlygreater for EM cases as the ln mean of 1.758±0.6618 was significantlygreater than in NE where the ln mean value was −0.8308±0.5155 (FIG.14A). Student's t test gave t=3.668 with p=0.0052, and the nullhypothesis that CD200 mRNA was not increased in EM was thereforerejected. For CD200R1, the ln mean was −2.1134±0.4844 compared to the NEln mean of −1.2638±0.8300. Student's t test gave t=0.904 for p=0.36 (2tail) for the null hypothesis that CD200R1 was not increased ordecreased. For CD200R2, however, the EM ln mean was −0.8390±0.6962 andthe NE ln mean was −5.1286±0.1484, and Student's t was 2.860 withp=0.0188 for the null hypothesis of no difference. The null hypothesisof no difference was rejected. Therefore, increased synthesis of CD200and CD200R2 was present in the secretory phase EM group compared to NEcontrols whereas a similar increase was not seen for CD200R1 mRNAlevels. The ratio of 2^(−ΔCt) values in the NE and EM group individualcases also fit a lognormal distribution. In the NE group, the R1/R2ratio ln mean±SEM was 3.3870±0.7063 compared to the EM group R1/R2 ratioln mean of −1.5624±0.7556. This difference was statistically significantby Student's t test (t=3.038, 9 df, p=0.0141 2 tail) allowing the nullhypothesis of no difference to be rejected and the alternativeconclusion that the reduction in the R1/R2 ratio in EM was not likelydue to chance to be accepted.

The ratio of CD200R1/CD200R2 mRNA was significantly reduced in the EMgroup compared to NE group. The change in ratio with CD200R1↓ andCD200R2↑ in EM proliferative phase samples as shown in FIG. 14A combinedwith CD200L and CD200S signaling can explain polarization of M2macrophages that normally dominate in secretory phase endometrium toexhibit an M1 proinflammatory phenotype that is thought to beembryo-unfriendly and contribute to infertility (Vallve-Juanico et al.,2019).

FIG. 14B shows the % of CD200R that was CD200R1 and CD200R2 as a piediagram for NE and EM cases in the proliferative, secretory, andmenstrual phases of their cycle. The trend for CD200R to more CD200R1than CD200R2 was evident in all phases of the cycle in EM cases but onlyachieved statistical significance in the secretory phase.

Example 4—IHC for CD200S

Anonymized tissue blocks from the initial IHC study of secretory phasehysterectomy samples (NE1, NE2, EM3, EM4, and EM5 described previously)and peritoneal endometrioma deposits were fixed in 10% buffered formalinfor 24 hours at room temperature (22-23° C.), processed, and embedded inparaffin. Paraffin blocks were cut using a Leica CM2255 Microtome(Wetzlar, Germany) into 4 μm sections. Sections were affixed topositively charged slides and allowed to dry overnight at roomtemperature. Slides were dewaxed and hydrated on the automated LeicaBOND Rx stain. Antigen retrieval was conducted using epitope retrievalbuffer 2 (Leica). Slides were stained for CD200L using rabbitantigen-affinity purified antibody for AA 45-95 of the conical sequence(Antibodies Online ABIN761396), for CD200S using rabbit antigen-affinitypurified antibodies for AA 170-220 (Antibodies-online, ABIN3189⁶6), andrabbit anti-KLH antibody, also antigen-affinity purified(Antibodies-Online, ABIN401183), was used as a control. All antibodieswere diluted 1:200 in Power Vision IHC Super Blocker (Leica) prior tostaining. Slides were stained using the Bond Polymer Refine Detectionkit (Leica). Slides were dehydrated, mounted in Fisher Chemical PermountMounting Medium (Fisher Scientific, Hampton, NH) and digitally scannedand analyzed using Imagescope (Leica), and photographed at 200× and400×.

Results:

As shown in FIG. 15A, photomicrographs (200× magnification—scale bar 100nm) illustrate immunohistochemical staining of control (NE) andendometriosis (EM) secretory phase endometrium with a 1/200 dilution ofantibody to CD200S which reacts with Exon 4 determinants of CD200variant 1 (amino acids (AA) 170-220). The staining indicates that thisantibody does not react in our IHC procedure with full length CD200(isoforms a and b), as access to AA 170-220 appears to be prevented byAA coded by Exon 2 and proximal Exon 3 AA. AA 170-220 become exposedwhen CD200 is truncated by elimination of exon 2 and proximal exon 3 AAs32-73 of CD200 isoform a (variant 1) to form CD200S the transcription ofwhich begins at AA 74 (M, methionine). This is compatible with CD200Sbeing UniParc/UniProt F8W7G1, NM_001318826, NP_001305757 and distinctfrom the conventional longer molecule (CD200L).

To determine if there was a greater concentration of CD200S⁺ cells insecretory phase EM endometrium, 11-13 photomicrographs done at 400× wereevaluated as described above. The mean±SEM per unit volume (0.0335 mm³as described above) was NE1 26.0±2.2, NE2 57.8±8.1, EM3 67.9±11.5, EM480.5±8.8, EM5 83.6±6.5, and combining these results, NE was 41.3±5.1 andEM was greater at 77.4±4.8 with t=5.143 and p<0.01 (1 tail).*Scale bar100 nm.

The photomicrographs of FIG. 15B illustrate immunohistochemical stainingof peritoneal endometrioma deposits corresponding to the EM cases inFIG. 5 for presence of CD200S⁺ leukocytes. In EM5, arrows point to arare presence of positive cells. This contrasts with abundant CD200S⁺cells in the endometrium. Scale bar 100 nm.

Endometriosis deposits may also be found within the endometrium asadenomyosis as shown in FIG. 15C. Case EM5 had adenomyosis and IHCresult shows peri-myometrial and deep myometrial zones where CD200L⁺glandular epithelium and CD200S⁺ cells in adjacent stroma/myometrium andbasal endometrium where CD2001⁺ glands which are intruding into themyometrium have been stained. This shows that CD200L and CD200S areclearly in different cells. Scale bar 100 nm. The photomicrograph showsthat CD200S⁺ cells were scant in the glandular deposit in myometrium.

The pattern of staining of CD200S⁺ cells is compatible with them beinguterine NK cells or a subset of NK cells. This was confirmed by cellcomparison to the frequency of CD56⁺ NK cells using a Leica mouseanti-CD56 antibody and a control antibody to CD68 on human macrophages(data not shown). CD200S in uNK cells has not previously been described.This discovery has implications for diagnosis of EM, if CD200S can bedetected in serum by ELISA combined with elevated CD200L, that couldpredict likelihood that ongoing ectopic implants will occur with eachmenses. This provides evidence that anti-CD200S monoclonal antibody oran equivalent to block the initial inflammatory response required forimplantation of ectopic tissue in combination with monoclonalanti-CD200L or an equivalent to abrogate suppression of the woman'simmune system cells that are able to destroy ectopic tissue e.g.macrophages and NK cells.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Where a term in the present application is found to bedefined differently in a document incorporated herein by reference, thedefinition provided herein is to serve as the definition for the term.

REFERENCES

-   -   Allan, S. E., Crome, D, Q., Crellin, N. K., Passerini, L.,        Steiner, T. B., Bacchetta, R., et al., 2007. Activation-induced        FOXP3 in human T effector cells does not suppress proliferation        or cytokine production. International Immunol. 19, 345-354.    -   Basta, P., Majka, M., Jozwicki, W., Lukaszewska, E., Knafel, A.,        Grabiec, M., et. al., 2010. The frequency of CD25+CD4+ and        FOXP3+ regulatory T cells in ectopic endometrium and ectopic        decidua. Reprod. Biol. Endocrinol. 8, 116.    -   Berbic, M., Hey-Cunningham, A. J., Ng, C., Tokushige, N.,        Ganewatta, S., Markham, R., et al., 2010. The role of Foxp3+        regulatory T-cells in endometriosis: a potential controlling        mechanism for a complex chronic condition. Human Reprod. 25,        900-907.    -   Berbic, M., Fraser, I. S., 2011 Regulatory T cells and other        leukocytes in the pathogenesis of endometriosis. J. Reprod.        Immunol. 88, 149-155.    -   Bokor, A., Debrock, S., Drijkoningen, M., Goossens, W., Fulop,        V., D'Hooghe, T. D., 2009. Quantity and quality of retrograde        menstruation: a case control study. Reprod. Biol. Endocrinol. 7,        123.    -   Brosens, I., Brosens, J. J., Benagiano, G., 2012. The eutopic        endometrium in endometriosis: are the changes of clinical        significance? Reprod. Biomed. Online 24, 496-502.    -   Budiu, R. A., Diaconu, I., Chrissluis, R., Drici, A.,        Edwards, R. P., Vlad, A. M., 2009. A conditional mouse model for        human MUC 1-positive endometriosis shown the presence of        anti-MUC1 antibodies and Foxp3+ regulatory T cells. Disease        Models and Mechanisms 2, 593-603.    -   Clark, D. A., 2016a. The importance of being a regulatory T cell        in pregnancy. J. Reprod. Immunol. 116: 60-69.    -   Clark, D. A., 2016b. Mouse is the new woman? Translational        research in reproductive immunology. Seminars in Immunopathology        38, 651-668.    -   Clark., D. A., Gorczynski, R. M., 2013. Immunological        tolerance/acceptance of the semiallogeneic embryo: decidual        transforming growth factors and tolerance signaling molecules.        In: G. Chaouat, G., Olivier, S., N Ledee, N. (Eds.), Immunology        of Pregnancy 2013, Bentham eBooks (eISBN: 978-1-60805-733-7),        pp. 540-558.    -   Clark, D. A., Dmetrichuk, J. M., McCready, E., Dhesy-Thind, S.,        Arredondo, J. L., 2017. Changes in expression of the CD200        tolerance-signaling molecule and its receptor (CD200R1) by        villus trophoblasts during first trimester misses abortion and        in chronic histiocytic intervillositis. Am. J. Reprod. Immunol.        78e12665, 1-7.    -   Chen Z, Chen D-X, Kai Y, Khatri I, Lamptey B, Gorczynski R M.        Identification of an expressed truncated form of CD200, CD200tr,        which is a physiological antagonist of CD200-induced        suppression. Transplantation 2008;86:1116-1124.    -   Clement, P. B., 2007. The pathology of endometriosis. A survey        of the many faces of a common disease emphasizing diagnostic        pitfalls and unusual and newly appreciated aspects. Adv. Anat.        Pathol. 14, 241-260.    -   Collette, T., Bellehumeur, C., Kats, R., Maheux, R., Maillous,        J., Villeneuve, M., Akoum., 2004. Evidence for an increased        release of proteolytic activity by eutopic endometrial tissue in        women with endometriosis and for involvement of matric        metaloproteinase-9. Human Reprod. 19, 1257-1264.    -   Dalbeth, N., Gundle, R., Davies, R. J., Lee, Y. C.,        McMichael, A. C., Callan, M. F., 2004. CD56^(bright) NK cells        are enriched at inflammatory sites and can engage with monocytes        in a reciprocal program of activation. J. Immunol. 173,        6418-6426.    -   Ferreira, V. H., Dizzell, S., Nazli, A., Kalka, J. K., Mueller,        K., Nguyen, P. V., et al., 2015. Medroxyprogesterone acetate        regulates HIV-1 uptake and transcytosis but not replication in        primary genital epithelial cells, resulting in enhanced T-cell        infection. JID 211, 1745-1756.    -   Fraser, I. S., McCatton, G., Markham, R., Resta, T., Watts,        A., 1986. Measured menstrual blood loss in women with        menorrhagia associated with pelvic disease or coagulation        disorders. Obstet. Gynaec. 61, 109-112.    -   Gargett, G. E., Schwab, K. E., Brosens, J. J., Puttemans, P.,        Benagiano, G., Brosens, I., 2014. Potential role of endometrial        stem/progenitor cells in the pathogenesis of early-onset        endometriosis. Molec. Hum. Reprod. 20, 591-598.    -   Gorczynski, R., Chen, Z., Kai, Y., Lee, L., Wong, S.,        Marsden, P. A., 2004. CD200 is a ligand for all members of the        CD200R family of immunoregulatory molecules. J. Immunol., 172,        7744-7749.    -   Hatherley, D., Cherwinski, H. M., Moshref, M., Barclay, A.        N., 2005. Recombinant CD200 protein does not bind activating        proteins closely related to CD200 receptor. J. Immunol. 175,        2469-2474.    -   Jetten, N., Verbruggen, S., Gijbels, M. J., Post, M. J., De        Winther M. P. J., Donners, M. M. P. C., 2014. Anti-inflammatory        M2, but non pro-inflammatory M1 macrophages promote angiogenesis        in vitro. Angiogenesis 17, 109-118.    -   Jones, R. K., Bulmer, J. N., Searle, R. F., 1996.        Immunohistochemical characterization of stromal leukocytes in        ovarian endometriosis: comparison of eutopic and ectopic        endometrium with normal endometrium. Fertil. Steril. 66, 81-89.    -   Kobyashi K, Yano H, Umakoshi A, Matsumoto S, Mise A, Funahashi        Y, Ueno Y, Kamei Y, Takada Y, Kumon Y, Olnishi T, Tanaka J. A        truncated form of CD200 (CD200S) expression on glioma cells        prolonged survival in a rat glioma model by induction of a        dendritic cell-like phenotype in tumor associated macrophages.        Neoplasia 2016;18:229-241.    -   Kos, 0., Hughson, R. L., Hart, D. A., Clement, G.,        Frings-Meuthen, P., Linnarsonm D., et al., 2014. Elevated serum        soluble CD200 and CD200R as surrogate markers of bone loss under        bed rest conditions. Bone 60, 33-40.    -   Kalu, E., Sumar, N., Giannopoulos, T., Patel, P., Croucher, C.,        Sherriff, E., et al., 2007. Cytokine profiles in serum and        peritoneal fluid from infertile women with and without        endometriosis. J. Obstet. Gynecol. Res. 33, 490-495.    -   Kaushic, C., Nazli, A., Ferreira, V. H., Kafka, J. K., 2011.        Primary human epithelial cell culture system for studying        interactions between female upper genital tract and sexually        transmitted viruses, HSV-2 and HSV-1. Methods 55, 114-121.    -   Kruitwagen, R. F. P. M., Pods, L. G., Willemsen, W. N. P., de        Ronde, I. J. Y., Jap, P. H. K., Roland, R., 1991. Endometrial        epithelial cells in peritoneal fluid during the follicular        phase. Fertil. Steril. 55, 297-303.    -   Li, M. Q., Wang, Y., Chang, K. K., Meng, Y. H., Liu, L. B., Mei,        J., et al., 2014. CD4⁺Foxp3⁺ regulatory T cell differentiation        mediated by an endometrial stromal-cell TECK promotes the growth        and invasion of endometrial lesions. Cell Death & Disease 5,        e14356.    -   Liu, K., Zhang, W., Liu, S., Dong, B., Liu, Y., 2015. Hepatic        endometriosis: a rare case and review of the literature.        Europ. J. Med. Res. 20, 48.    -   Luo, Q., Ning, W., Wu, Y., Zhu, X., Jin, F., Sheng, J., et        al., 2006. Altered expression of interleukin-18 in the ectopic        and eutopic endometrium of women with endometriosis. J. Reprod.        Immunol. 72, 108-117.    -   Matsuzaki, S., Canis, M., Pouly, J. L., Botchorishvili, R.,        Deschelotte, P. J., Mage, G., 2006. Differential expression of        genes in eutopic and ectopic endometrium from patients with        ovarian endometriosis. Fertil. Steril. 86, 548-553.    -   Mbarik, M., Kaabachi, W., Henidi, B., Sassi, F. H., 2015.        Soluble ST2 and IL-33: Potential markers of endometriosis in the        Tunisian population. Immunol. Letters 166, 1-5.    -   Mei, J., Jin, L. P., Ding, D., Li, M. Q., Zhu, X. Y., 2012.        Inhibition of IDO1 suppresses cycloxygenase-2 and matrix        metalloproteinase-9 expression and decreases proliferation,        adhesion and invasion of endometrial stromal cells. Molec, Human        Reprod. 18, 467-476.    -   Mei, J., Li, M. Q., Ding, D., Li, D. J., Jin, L. P., Hu, W. G.,        Zhu, X. Y., 2013. Indoleamine 2,3-dioxygenase-1 (IDO1) enhances        survival and invasiveness of endometrial stromal cells via the        activation of the JNK signaling pathway. Int. J. Clin. Exp.        Pathol. 6, 431-444.    -   Mei, J., Zhu, X Y., Jin, L P., Duan, Z L., Li, D J., Li,        M-Q., 2015. Estrogen promotes the survival of human secretory        phase endometrial stromal cells via CXCL12/CXCR4        up-regulation-mediated autophagy inhibition. Human Reprod. 30,        1677-1689.    -   Provinciali, M., Di Stefano, G., Muzzioli, M., Garzetti, G. G.,        Ciavattini, A., Fabris, N., 1995. Relationship between        17-β-estradiol and prolactin in the regulation of natural killer        cell activity during progression of endometriosis. J.        Endocrinol. Invest. 18, 645-652.    -   Ridley, J. H., Edwards, K., 1958. Experimental endometriosis in        the human. Am. J. Obstet. Gynecol. 76, 783-789.    -   Scott, R. B., Te Linde, R. W., 1954. Further studies on        experimental endometriosis. Am. J. Obstet. Gynecol. 66,        1082-1099.    -   Smith, K. A., Pearson, C. B., Hachey, A. M., Xia, D-L.,        Wachtman, L. M., 2012. Alternative activation of macrophages in        rhesus macaques (Macaca mulatta) with endometriosis. Comparative        Medicine 62, 303-310.    -   Somigliana, E., Vigano, P., Gaffuri, B., Candiani, M., Busacca,        M., Di Blasio, A. M., et al., 1999. Modulation of NK cell lytic        function by endometrial secretory factors: potential role in        endometriosis. Am. J. Reprod. Immunol. 36, 295-300.    -   Somigliana, E., Candiana, M., Vignali, M., Vigano, P., 2001.        Impaired natural killer cell activity in endometriosis?—A        technical challenge for validation. Fertil. Steril. 76, 422.    -   Te Linde, R. W., Scott, R. B., 1950. Experimental endometriosis.        Am. J. Obstet. Gynecol. 60, 1147-1166.    -   Tirado-Gonzalez, I., Barrientos, G., Tariverdian, N., Arck, P.        C., Garcia, M. G., Klapp, B. F., et al., 2010. Endometriosis        research: animal models for the study of a complex disease. J.        Reprod. Immunol. 86, 141-147.    -   Treloar, S. A., O'Connor, D. T., O'Connor, V. M., Martin, N.        G., 1999. Genetic influence on endometriosis in an Australian        twin sample. Fertil Steril. 71, 701-710.    -   Vallve-Juanico J, Santamaria X, Vo K C, Houshdaran S, Giudice        L C. Macrophages display proinflammatory phenotypes in the        eutopic endometrium of women with endometriosis with relevance        to an infectious etiology of the disease. Fertin Steril        2019;112:1118-1128.    -   Vigano, P., Vercellini, P., Di Blasio, A. M., Colombo, A.,        Candiani, G. B., Vignali, M., 1991. Deficient antiendometrium        lymphocyte-mediated cytotoxicity in patients with endometriosis.        Fertil. Steril. 56, 894-899.    -   Vigano, P., Somigliana, E., Vignali, M., Busacca, M., Blasio, A.        M., 2007. Genetics of endometriosis: current status and        prospects. Front. Biosci. 12, 3247-3255    -   Wessels J M, Kay V R, Leyland N A, Agarwal S K, Foster        W G. 2016. Assessing brain-derived neurotrophic factor as a        novel clinical markers in endometriosis. Fertil. Steril.        105:119-28. Woidacki, K., Meyer, N., Schumacher, A.,        Goldschmidt, A., Maurer, N., Zenclussen, A. C., 2015. Transfer        of regulatory T cells into abortion-prone mice promotes the        expansion of uterine mast cells and normalizes early pregnancy        angiogenesis. Sci. Rep. 5, 13938    -   Wolf, G. C., Singh, K. B., 1989. Caesarian scar endometriosis: a        review. Obstet. Gynecol. Survey 44, 89-95.    -   Wong, K. K., Khatri, I., Shaha, S., Spanner, D. E.,        Gorczynski, R. M., 2010. The role of CD200 in immunity to B cell        lymphoma. J. Leuk. Biol. 88, 361-372    -   Wong, K. K., Brennerman, F., Chesney, A., Spaner, D. E.,        Gorczynsiki, R. M., 2012. Soluble CD200 is critical to engraft        chronic lymphocytic leukemia cells in immunocompromised mice.        Cancer Res. 72, 4931-4943.    -   Wong, K. K., Zhu, F., Khatri, I., Huo, Q., Spaner, D. E.,        Gorczynski, R. M., 2016. Characterization of CD200 ectodomain        shedding. PLoS ONE 11, e0152073.

Wright, G. J., Cherwinski, H., Foster-Cuevas, M., Brooke, G., Puklavec,M. J., Bilger, M., et al., 2003. Characterization of the CD200 receptorfamily in mice and humans and their interaction with CD200. J. Immunol.171, 3034-3046.

-   -   Wu, M., Yang, J., Chao, K., Hwang, J., Yang, Y., Ho, H., 2000.        Increase in expression of killer inhibitory receptors on        peritoneal natural killer cells in women with endometriosis.        Fertil. Steril. 74, 1187-1191.    -   Xu, H., Zhang, T., Man, G. C. W., May, K. E., Becker, C. M.,        Davis, T. N., et al., 2013. Vascular endothelial growth factor C        is increased in endometrium and promotes endothelial functions,        vascular permeability and angiogenesis and growth of        endometriosis. Angiogenesis 16, 541-551.    -   Yu, J. H., Lin, X. Y., Wang, L., Liu, Y., Fan, C. F., Zhang, Y.,        et al., 2013. Endobronchial endometriosis presenting as        central-type lung cancer: a case report. Diagnostic Pathol. 8,        53.    -   Zamah, N. M., Dodson, M. G., Stephens, L. C., Buttram, V. C.        Jr., Besch, P. G., Kaufman, R. H., 1984. Transplantation of        normal and ectopic endometrial tissue into athymic nude mice.        Am. J. Obstet. Gynecol. 149, 591-597.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. A method of monitoring a mammal followingtreatment of endometriosis comprising: i) detecting the level ofsecretory phase CD200L in a biological sample from the mammal using amethod which does not detect CD20S; ii) comparing the CD200L level to apre-treatment CD200L control level; and iii) determining that the mammalis responding to treatment when the CD200L level is reduced incomparison to the control level.
 9. A kit for use in a method as definedin claim 5 comprising a CD200L-specific reactant and optionallyinstructions for use in the method.
 10. The kit of claim 9, wherein theCD200L-specific reactant is an antibody that targets amino acids encodedby exon 2, or one or more nucleotide primers that bind to exon
 2. 11.The kit of claim 10, wherein the reactant is associated with anindicator that yields a detectable signal when the reactants bind toCD200L.
 12. (canceled)
 13. (canceled)
 14. (canceled)
 15. (canceled) 16.(canceled)
 17. A method of diagnosing endometriosis in a female subject,the method comprising: a) detecting the level of CD200L in a secretoryphase biological sample from the subject using a method which does notdetect CD200S; b) detecting the level of CD200S in the biologicalsample; b) comparing the level of CD200L in the biological sample withthe level of CD200S; and c) diagnosing the subject with endometriosiswhen the level of CD200L in the biological sample is higher than thelevel of CD200S in the sample.
 18. A method of identifying risk ofinfertility in a female mammal comprising the steps of: a) detecting thelevel of CD200R1 and CD200R2 in a biological sample from the mammal; b)comparing the level of CD200R1 to CD200R2 in the biological sample; andc) identifying that the mammal is at risk of infertility when the levelof CD200R2 is higher than the level of CD200R1 in the biological sample.19. The method of claim 18, wherein the biological sample is aproliferative; secretory or menstrual phase sample.
 20. The method ofclaim 19, wherein the biological sample is a secretory phase sample.